The long-term goal is to determine the mechanism by which mesenchymal stem cells differentiate into osteoblasts. This goal is central to implementing various tissue- and gene- based therapies for osteoporosis, rheumatic diseases, developmental bone defects and severe fractures. This research plan will test two candidate intracellular signals to determine how they modulate the differentiation of murine stem cells into osteoblasts. The first candidate is the apoptosis inhibitor, Bcl-2. The first hypothesis is that enforced bcl-2 expression in stem cells and osteoprogenitors will result in a higher osteoprogenitor frequency, while the absence of Bcl-2 will result in a lower osteoprogenitor frequency. The second candidate is the osteoblast-specific transcription factor Cbfa1. The second hypothesis is that enforced cbfa1 expression in stem cells and osteoprogenitors will result in a higher osteonrogenitor frequency, while the absence of cbfa1 will result in the absence of the osteoblast lineage. The experiments quantify mineralized bone nodule formation and gene expression characteristic of the osteoblast lineage. Two different sources of murine stem cells will be utilized, bone marrow and embryonic stem cells. Differentiation of embryonic stem cells into osteoblasts represents a new avenue of generating osteoblasts in vitro and will facilitate the study of genetic alterations that impact differentiation, proliferation and function of the osteoblast lineage.